Intestinal barrier function is compromised in inflammatory, infectious, ischemic, and immune-mediated intestinal disease. In the previous cycle we studied mechanisms and impact of tumor necrosis factor- (TNF-) induced barrier loss using in vitro and in vivo models to: i) define TNF-induced, myosin light chain kinase- (MLCK-) dependent tight junction regulation; ii) show that MLCK is required for TNF-induced diarrhea; iii) identify and characterize the human MLCK promoter; iv) document MLCK activation in human inflammatory bowel disease (IBD); and v) show that chronic epithelial MLCK activation stimulates mucosal immune cells and sensitizes mice to experimental IBD. We have now begun to define the mechanisms by which TNF specifically activates MLCK1, a long MLCK splice variant, to trigger endocytosis of the tight junction protein occludin. This creates tight junction 'leaks' that allow paracellular flux of large macromolecules without affecting ion selectivity. Our preliminary data show that expression of constitutively-active MLCK in vivo increases flux across the 'leak' pathway. Moreover, unlike in vitro MLCK activation, chronic in vivo MLCK activation alters ion selectivity of the paracellular barrier. This is due to increased mucosal IL-13 production, which induces epithelial claudin-2 expression to create small, cation-selective 'pores' that allow Na+, but not large macromolecules, to traverse the paracellular pathway. This in vivo result emphasizes the presence of, as well as interactions between, two distinct pathways across the tight junction; a high capacity 'pore' pathway that allows small uncharged solutes and specific ions to pass and a low capacity 'leak' pathway that is permeable to larger, uncharged macromolecules but is not ion selective. The fact that 'pore' and 'leak' pathways interact through the mucosal immune system, i.e. IL-13 production, suggests that these pathways may make distinct contributions to disease pathogenesis. These and other data have led to the central hypothesis that at least two mechanisms of regulation modulate unique paracellular pathways, i.e. 'pore' and 'leak,' to differentially impact mucosal homeostasis and disease pathogenesis. The aims of this application are to i) define the mechanisms by which TNF triggers MLCK1 perijunctional trafficking and enzymatic activation to enhance flux across the tight junction 'leak' pathway; ii) identify the mechanisms that regulate claudin-2 expression and 'pore' pathway flux; and iii) determine the impact of MLCK, occludin, and claudin-2 expression on 'pore' and 'leak' pathway function as well as initiation and progression of chronic intestinal disease. As a whole, these studies will define the relative roles of specific epithelial regulatory processes and immune signaling in modulation of 'pore' and 'leak' pathway paracellular flux and disease pathogenesis. The data, which will provide new understanding of the mechanisms by which intestinal barrier function contributes to human health and disease, will lay the foundation necessary for development of novel therapeutic strategies to correct barrier dysfunction. PUBLIC HEALTH RELEVANCE: The intestinal lining (epithelium) must maintain a barrier that keeps the intestinal contents separate from the remainder of the body. This function is frequently compromised in intestinal disease and has been implicated as an early step in disease development. The proposed studies will advance mechanistic understanding of barrier regulation and dysregulation and, therefore, lead to development of novel therapeutic approaches to improve human health.